A normal host immune response requires the precise regulation of cytokine genes by T lymphocytes. Beneficial immune response to infectious agents or cancer, as well as deleterious responses observed in autoimmune diseases or allergy, are associated with the differentiation of helper T cells into distinct populations defined by differences in the expression of cytokine genes. Thus, the rational development of therapies to modulate the immune responses will require a detailed understanding of how these genes are differentially expressed. Analyses of cytokine gene regulation have identified several families of transcription factors that translate intracellular of cytokine gene regulation have identified several families of transcription factors that translate intracellular signaling events that defined transcriptional response. How these proteins mediate the differential regulation of signaling events into defined transcriptional responses. How these proteins mediate the differential regulations of gene expression that occurs during TH differentiation, however, remains unclear. Recent studies indicate that NFAT transcription factors in particular play a key role in this process. However, since multiple NFAT proteins with overlapping DNA specifies are co-expressed in T cells, determining the role of distinct family members in regulating TH differentiation has been difficult. Furthermore, in vivo studies of NFAT function in transgenic and "knockout" mice highlight significant gaps in our understanding of NFAT-dependent transcriptional control. In order to further define the transcriptional regulatory mechanisms involved in TH differentiation, we propose the following specific aims: In Aim #1, studies will focus on the sole NFAT family member that is induced upon T cell activation, NFAT2/c. The hypothesis that the NFAT2/c gene is subject to differential transcriptional regulation during TH differentiation will be tested by identify the proteins that regulate the NFAT2/c gene and determining how these proteins are regulated. In Aim #2m the role played by NFAT5 in regulating TH differentiation will be determined. NFAT5 is a newly identified NFAT protein that is also an inducible early response gene in T cells. The hypothesis that NFAT5 represents an important signal-targeted regulator of T cell gene expression and differentiation will be tested through studies of NFAT5 expression, biochemical modification, and function. In Aim #3, combinatorial transcriptional control by NFAT proteins will be further defined by testing the hypothesis that the discrepancy between the highly similar in vitro DNA binding specificity of NFAT proteins and their distinct in vivo specificity results from association with additional nuclear co-factors. Finally, in Aim #4, a novel expression cloning approach will be taken to test the hypothesis that defined transcriptional repressors or silencers play an important role in regulating TH differentiation.